Yellow rust (YR) globally affects wheat crops. It may turn into an epidemic, resulting in significant yield losses if the environment is suited to YR spread. The provision of resistant wheat cultivars is a sustainable protection strategy against YR. The current study aimed to use a combination of classical analytical tools to identify potential wheat lines through screening under YR stress for utilization in YR-resistance breeding. A total of 14 parents, which included 9 lines and 5 testers, were formed into 45 cross combinations via line × tester mating format. The tested germplasm was grown in a triplicate randomized full-block design, under optimal and yellow rust (YR)-stress conditions. Data were recorded on different morphological, physiochemical, yield and component traits at appropriate crop stages. A pre-combining ability analysis revealed significant inter-genotype variations. A combining ability study identified non-additive gene action in the inheritance of most of the investigated traits. Four potential parents (PR128, AN179, KS17 and WD17) and two crosses (PR128 × WD17 and AN179 × KS17) retained higher combining ability values for yield traits under YR-stress. A cluster analysis based on the overall performance found divergent classes among the screened genotypes. The clustering of different genotypes was shifted under YR-stress, which suggests variable genotypic response. Through factor analysis, we assessed and confirmed genotypes performing consistently under YR-stress. The identified genotypes may be used for disease-resistance breeding in wheat. Based on their positive correlation with grain yield, we suggest the use of peduncle length and tillers per plant as phenotypic markers for wheat selection and breeding. The knowledge base generated through the current study will add to the ongoing research on sustainable wheat breeding program.